Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC-EVs) have been proposed as a novel therapeutic tool with numerous clinically related advantages. However, their characteristics and functionality are dependent on the source of MSCs and their cell culture conditions. Fetal bovine serum (FBS) provides a source of nutrients and growth factors to the cultured cells. However, certain pitfalls are associated with its supplementation to the culture media, including introduction of exogenous FBS-derived EVs to the cultured cells. Thus, recent practices recommend utilization of serum-free (SF) media or EV-depleted FBS. On the contrary, evidence suggests that the immunomodulatory ability of MSC-EVs can be improved by exposing MSCs to an inflammatory (IF) environment. The objective of this study was to (1) compare EVs isolated from two tissue sources of MSCs that were exposed to various cell culture conditions and (2) to evaluate their anti-inflammatory effects. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) and umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were exposed to either a SF media environment, an IF environment, or media supplemented with 5% EV-depleted FBS. Following isolation of MSC-EVs, the isolates were quantified and evaluated for particle size, phenotypic changes, and their immunomodulatory potential. A statistically significant difference was not identified on the yield and protein concentration of different isolates of EVs from BM-MSCs and UC-MSCs, and all isolates had a circular appearance as evaluated via electron microscopy. A significant difference was identified on the phenotype of different EVs isolates; however, all isolates expressed classical markers such as CD9, CD63, and CD81. The addition of BM-derived MSC-EVs from FBS environment or UC-derived MSC-EVs from IF environment resulted in statistically significant downregulation of IL-6 messenger RNA (mRNA) in stimulated leukocytes. This study confirms that EVs produced by different MSC sources and cell culture conditions affect their phenotype and their immunomodulatory capacities.

Objectives Phage–gonadotropin-releasing hormone (GnRH) constructs with potential contraceptive properties were generated in our previous study via selection from a phage display library using neutralizing GnRH antibodies as selection targets. In mice, these constructs invoked the production of antibodies against GnRH and suppressed serum testosterone. The goal of this study was to evaluate this vaccine against GnRH for its potential to suppress reproductive characteristics in cats. Methods Sexually mature male cats were injected with a phage–GnRH vaccine using the following treatment groups: (1) single phage–GnRH vaccine with adjuvant; (2) phage–GnRH vaccine without adjuvant and half-dose booster 1 month later; or (3) phage–GnRH vaccine with adjuvant and two half-dose boosters with adjuvant 3 and 6 months later. Anti-GnRH antibodies and serum testosterone, testicular volume and sperm characteristics were evaluated monthly for 7–9 months. Results All cats developed anti-GnRH antibodies following immunization. Serum antibody titers increased significantly after booster immunizations. In group 3, serum testosterone was suppressed 8 months after primary immunization. Total testicular volume decreased in group 1 by 24–42% and in group 3 by 15–36% at 7 months after immunization, indicating potential gonadal atrophy. Vacuolation of epididymides was observed histologically. Although all cats produced sperm at the conclusion of the study, normal morphology was decreased as much as 38%. Phage alone produced no local or systemic reactions. Immunization of phage with AdjuVac produced unacceptable injection site reactions. Conclusions and relevance Our phage-based vaccine against GnRH demonstrated a potential for fertility impairment in cats. Future research is required to optimize vaccine regimens and identify animal age groups most responsive to the vaccine. If permanent contraception (highly desirable in feral and shelter cats) cannot be achieved, the vaccine has a potential use in zoo animals or pets where multiple administrations are more practical and/or reversible infertility is desirable.

Overpopulation of free-roaming and wildlife animals negatively affects economy and public health in many parts of the world. Contraceptive vaccines are viewed as a valuable option for reducing numbers of unwanted animals. This study develops vaccines for potential use in animal contraception exploiting a DNA platform. Objectives of the study were to generate DNA constructs directed against gonadotropin-releasing hormone receptor (GnRHR), a crucial molecular player in animal reproduction, and characterize them for ability to promote immune responses and suppression of reproductive parameters in vivo. DNA constructs were created to encode for a recombinant protein composed of two domains: GnRHR, the target antigen, and ubiquitin (Ub), a support protein. Ub-GnRHR constructs administered intramuscularly or intradermally or containing different promoters were compared. CMV and EF1α promoters were shown to be superior to CAG. In fertility trials, mice immunized intradermally with Ub-GnRHR construct driven by EF1α had a significantly lower number of fetuses. Importantly, the impaired fertility was achieved with a single DNA immunization and without the use of adjuvants. The study demonstrated for the first time that targeting the GnRH receptor with DNA-based vaccines could be a viable option for animal contraception.

Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5 × 1011 virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2 × 1012 vir per mouse in combination with a poly(lactide-co-glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems.

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76 °C for 3 h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines.

Multiple phage-peptide constructs, where the peptides mimic sperm epitopes that bind to zona pellucida (ZP) proteins, were generated via selection from a phage display library using a novel approach. Selections were designed to allow for identification of ZP-binding phage clones with potential species-specific properties, an important feature for wildlife oral vaccines as the goal is to control overpopulation of a target species while not affecting non-target species’ reproduction. Six phage-peptide antigens were injected intramuscularly into pigs and corresponding immune responses evaluated. Administration of the antigens into pigs stimulated production of anti-peptide antibodies, which were shown to act as anti-sperm antibodies. Potentially, such anti-sperm antibodies could interfere with sperm delivery or function in the male or female genital tract, leading to contraceptive effects. Staining of semen samples collected from different mammalian species, including pig, cat, dog, bull, and mouse, with anti-sera from pigs immunized with ZP-binding phage allowed identification of phage-peptide constructs with different levels of species specificity. Based on the intensity of the immune responses and specificity of these responses in different species, two of the antigens with fusion peptide sequences GEGGYGSHD and GQQGLNGDS were recognized as the most promising candidates for development of contraceptive vaccines for wild pigs.

Zona pellucida (ZP) glycoproteins play a central role in sperm–oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs.

The liver and lung are not only described as "target organs" in sepsis in most species, but are purported to be sources of circulating inflammatory mediators central to the systemic inflammatory response syndrome (SIRS). As we have recently reported an inflammatory response in the laminar tissue in laminitis similar to that described in "target organs" in human sepsis, we investigated the inflammatory response of the lung and liver in the black walnut extract (BWE) model of equine laminitis to determine (1) if a similar systemic inflammatory response occurs in this laminitis model as described for these organs in human sepsis, and (2) if these organs may be an important source of the inflammatory mediators leading to laminar inflammation. Real-time quantitative PCR (RT-qPCR) was used to measure hepatic and pulmonary mRNA concentrations of IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha, COX-1 and COX-2. Hepatic samples were assessed from two time points in the developmental/prodromal period: (1) 1.5h post-BWE administration (BWE-1.5H, n = 5), and (2) the "developmental time point" (onset of leukopenia, approximately 3h post-BWE administration, BWE-DEV, n = 5). Pulmonary samples were only assessed for the BWE-DEV group. One control group (CON-3H, n = 5) was used for both the 1.5H and DEV groups. Finally, CD13 immunohistochemistry was performed to assess leukocyte emigration into hepatic and pulmonary parenchyma. Hepatic and pulmonary mRNA concentrations of the proinflammatory cytokines IL-6, IL-8 and TNF-alpha were significantly increased (P < 0.05) in BWE-1.5H and BWE-DEV groups compared to the control group; IL-1beta mRNA concentrations were only increased in the lung. The "anti-inflammatory" cytokines, IL-10 and IL-4, underwent transient decreases at different time points. Significant increases in parenchymal leukocyte numbers occurred in both the lung and liver at the BWE-DEV time point. Hepatic and pulmonary proinflammatory cytokine expression differ from that previously reported for the laminae in that TNF-alpha was increased in the hepatic and pulmonary tissues, the increases in expression of IL-6 and IL-8 are dramatically smaller for the liver and lung compared to those reported for the laminae, and the peak changes appear to occur later in the disease process in the liver than in the laminae (BWE-DEV in liver vs. 1.5H in the laminae).

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.

The objective of this research was to evaluate reactivation of bovine viral diarrhea virus (BVDV) following dexamethasone treatment in 4 bulls that had previously been inoculated with BVDV, 3 of which had been demonstrated to have a localized testicular infection. Bulls were housed in an isolated pasture with in-contact steers. Beginning on day 0 of this study, all bulls received a daily dose of 0.1 mg/kg body weight (BW) of dexamethasone intravenously for 5 consecutive days. Blood was collected from the in-contact steers and semen, blood, and cerebrospinal fluid were collected from the bulls during and following dexamethasone treatment. Samples were assayed for BVDV using virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR). Serum was assayed for antibody using standard virus isolation. Virus was not isolated from blood, cerebrospinal fluid, or semen from any of the 4 bulls during the study period. One of the bulls was positive for BVDV in semen by RT-nPCR throughout the study period. The BVDV was not recovered from any in-contact control steers during the 28-day study period, nor did any of the in-contact control steers seroconvert to BVDV. Raw semen from 1 bull that was RT-nPCR positive was intravenously inoculated into 7 seronegative steers based upon the Cornell Semen Test. The BVDV could not be recovered from the steers and none of them seroconverted to BVDV. The results indicated that reactivation of BVDV in bulls with a localized testicular infection is unlikely; however, further research is necessary to determine the full potential for BVDV transmission from bulls with a localized testicular infection.

BACKGROUND:Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis.HYPOTHESIS:Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis.ANIMALS:Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n = 5), CON-3h (control group for DEV, n = 5), LAM (n = 5) and CON-10h (control group for LAM, n = 5).METHODS:Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2.RESULTS:Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group.CONCLUSIONS AND CLINICAL IMPORTANCE:COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.

The objectives of this research were to evaluate the risk of prolonged testicular infection as a consequence of vaccination of peri-pubertal bulls with a modified-live, noncytopathic strain of BVDV and to assess vaccine efficacy in preventing prolonged testicular infections after a subsequent acute infection. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with an approximate minimum immunizing dose or a 10× standard dose of modified-live, noncytopathic BVDV or were maintained as unvaccinated controls. Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry and the identity of viral strains was determined by nucleotide sequencing of PCR products. The vaccine strain of BVDV was detected in testicular tissue of vaccinated bulls as long as 134 days after immunization. Prolonged testicular infections with the challenge strain were detected only in unvaccinated bulls as long as 85 days after challenge. Whereas vaccination caused prolonged testicular infection in some bulls, it did prevent subsequent infection of testicular tissue with the challenge strain. This research demonstrates that subcutaneous vaccination of naïve, peri-pubertal bulls with a noncytopathic, modified-live strain of BVDV can result in prolonged viral replication within testicular tissue. The risk for these prolonged testicular infections to cause venereal transmission of BVDV or subfertility is likely to be low but requires further investigation.